HYBRIDS AND PHYLOGENETIC SYSTEMATICS I. PATTERNS OF CHARACTER EXPRESSION IN HYBRIDS AND THEIR IMPLICATIONS FOR CLADISTIC ANALYSIS

Interspecific hybridization is considered common among plants, but the methods of cladistic systematics produce only divergently branching phylogenetic hypotheses and thus cannot give the correct phylogeny if an analysis includes hybrids. Empirical studies of the impact of known hybrids on phylogenetic analysis are lacking, and are necessary to begin to understand the problems that we face if hybrids are often included in cladistic analysis. Examination of the implications of hybrids for cladistics must begin with patterns of character expression in hybrids. This study includes 17 hybrids and their nine parental taxa that are Central American species of Aphelandra (Acanthaceae), analyzed using a set of 50 morphological characters. The hybrids are overwhelmingly intermediate as quantitatively scored for phylogenetic analysis. They express maternal and paternal, and primitive and derived characters in equal frequencies, showing no evidence of predominant inheritance of derived character states as has been assumed by most cladists who have considered hybrids theoretically. Because of their known genetic constitution, hybrids were useful in homology assessment and ordering character states. The parental character set was generally robust, but some changes were made to reflect the special evidence offered by the hybrids. These hybrids suggest that the inclusion of hybrids in phylogenetic analysis will not lead to unresolved cladograms with rampant homoplasy, as has been predicted by other authors. Instead, the patterns of character inheritance in these hybrids lead to the prediction that a hybrid will be placed by phylogenetic analysis as a basal lineage to the clade that includes its most derived parent, with relatively little effect on homoplasy. These predictions will be evaluated by incorporation of the hybrids in phylogenetic analyses, to be reported in a subsequent paper.     

A novel ontogenetic pathway in hybrid embryos between species with different modes of development

To investigate the bases for evolutionary changes in developmental mode, we fertilized eggs of a direct-developing sea urchin, Heliocidaris erythrogramma, with sperm from a closely related species, H. tuberculata, that undergoes indirect development via a feeding larva. The resulting hybrids completed development to form juvenile adult sea urchins. Hybrids exhibited restoration of feeding larval structures and paternal gene expression that have been lost in the evolution of the direct-developing maternal species. However, the developmental outcome of the hybrids was not a simple reversion to the paternal pluteus larval form. An unexpected result was that the ontogeny of the hybrids was distinct from either parental species. Early hybrid larvae exhibited a novel morphology similar to that of the dipleurula-type larva typical of other classes of echinoderms and considered to represent the ancestral echinoderm larval form. In the hybrid developmental program, therefore, both recent and ancient ancestral features were restored. That is, the hybrids exhibited features of the pluteus larval form that is present in both the paternal species and in the immediate common ancestor of the two species, but they also exhibited general developmental features of very distantly related echinoderms. Thus in the hybrids, the interaction of two genomes that normally encode two disparate developmental modes produces a novel but harmonious ontongeny.     

Acoustic signal characters in interspecific hybrids of bank and red-backed voles

It has been found that distress signals of bank and northern red-backed voles and their hybrids do not differ in qualitative characters, but the modes of the acoustic signal parameters in many cases differ in the hybrids depending on their origin and can be correlated with the inheritance of some morphological characters that are typical for the species. It is noted that the distress-signal frequencies (quartiles) differ significantly in the first-generation hybrids of various origin and their characteristics are closer to those of the paternal parent species. It has been found that the inheritance of the frequency characteristics by the second-generation hybrids does not have a well-pronounced regularity.     

Inheritance of some quantitative morphological characters in reciprocal hybrids of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis>and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae)

Some quantitative characters of three-summer old reciprocal hybrids of beluga Acipenser huso and starred sturgeon A. stellatus,as well as of juveniles of their parental species, are comparatively analyzed. The found maternal and paternal effects in manifestation of some characteristics in hybrids are considered in connection with elucidation of inheritance type of morphological characters in acipenserids. The paternal effect observed by some characters in hybrids beluga × starred sturgeon is explained by prevalence of dominant alleles in the genome of starred sturgeon in comparison with beluga. The maternal effect is elucidated by a few characteristics related to parameters of the head, first of all to the volume of the brain region of skull. Evidently, in this case, the matrocliny depends on the fact that information on the number of segments of embryo and their position is determined by the maternal organism during oogenesis.     

Novel gene expression patterns in hybrid embryos between species with different modes of development

SUMMARY Cross-species hybrids between eggs of the direct-developing sea urchin, Heliocidaris erythrogramma , and sperm from its congeneric indirect-developing species, Heliocidaris tuberculata, show restoration of features of the paternal feeding pluteus larva, including the gut, and pluteus spicular skeleton. Unlike other reported sea urchin cross-species hybrids, Heliocidaris hybrids express genes derived from both maternal and paternal species at high levels. Ectodermal cell types, which differ radically between the two parental species, are of intermediate form in the hybrids. Gene expression patterns in hybrid embryo tissues represent a number of combinations of parental gene expression patterns: genes that are not expressed in one paternal species, but are expressed in hybrids as in the expressing parent; genes that show additive expression patterns plus novel sites of expression; a gene that is misexpressed in the hybrids; and genes expressed identically in both parents and in hybrids. The results indicate that both conserved and novel gene regulatory interactions are present. Only one gene, CyIII actin , has lost cell-type-specific regulation in the hybrids. Hybrids thus reveal that disparate parental genomes, each with its own genic regulatory system, can produce in combination a novel gene expression entity with a unique ontogeny. This outcome may derive from conserved gene regulatory regions in downstream genes of both parental species responding in conserved ways to higher-level regulators that determine modular gene expression territories.     

Regulatory punctuated equilibrium and convergence in the evolution of developmental pathways in direct-developing sea urchins

We made hybrid crosses between closely and distantly related sea urchin species to test two hypotheses about the evolution of gene regulatory systems in the evolution of ontogenetic pathways and larval form. The first hypothesis is that gene regulatory systems governing development evolve in a punctuational manner during periods of rapid morphological evolution but are relatively stable over long periods of slow morphological evolution. We compared hybrids between direct and indirect developers from closely and distantly related families. Hybrids between eggs of the direct developer Heliocidaris erythrogramma and sperm of the 4-million year distant species H. tuberculata, an indirect developer, restored feeding larval structures and paternal gene expression that were lost in the evolution of the direct-developing maternal parent. Hybrids resulting from the cross between eggs of H. erythrogramma and sperm of the 40-million year distant indirect-developer Pseudoboletia maculata are strikingly similar to hybrids between the congeneric hybrids. The marked similarities in ontogenetic trajectory and morphological outcome in crosses of involving either closely or distantly related indirect developing species indicates that their regulatory mechanisms interact with those of H. erythrogramma in the same way, supporting remarkable conservation of molecular control pathways among indirect developers. Second, we tested the hypothesis that convergent developmental pathways in independently evolved direct developers reflect convergence of the underlying regulatory systems. Crosses between two independently evolved direct-developing species from two 70-million year distant families, H. erythrogramma and Holopneustes purpurescens, produced harmoniously developing hybrid larvae that maintained the direct mode of development and did not exhibit any obvious restoration of indirect-developing features. These results are consistent with parallel evolution of direct-developing features in these two lineages.     

Inheritance of foliar and nodal anatomical characters in some Ilex hybrids

The diverse relationships of the main differential leaf and nodal anatomical characters of a number ot artificial Ilex hybrids and their parents are compared. The marginal sclerenchyma strands character is not transferred to hybrids when differential. The lignification of the unspecialized abaxial epidermal cells is dominantly inherited, except in one hybrid. The development of a distinctive adaxial hypodermis is usually suppressed in hybrids when one of its parents is lacking in this character. New characters are reported for hybrids of Ilex aquifolium × opaca , where the vasculation of petiole and midrib and the presence of periclinal subdivisions in the adaxial epidermis constitute features not known from either parent species.
The possible significance of the four different ways of expression of parental characters in the hybrid offspring is discussed with reference to: genetic control of anatomical characters; the identification of hybrid material; the evolution of leaf anatomical diversity in Ilex , and the systematic value of leaf anatomical characters.     

Morphology and gene analysis of hybrids between two congeneric sea stars with different modes of development

The sea star Astropecten scoparius has feeding bipinnarian larvae, whereas its congener Astropecten latespinosus has nonfeeding barrel-shaped larvae. To investigate evolutionary changes in the development of asteroids, we performed reciprocal crosses between these two species with different larval forms. In the cross between A. scoparius eggs and A. latespinosus sperm, embryos developed into bipinnaria-like larvae. The larvae exhibited either a functional digestive system (a maternal feature) or a nonfunctional digestive system with the tip of the archenteron not connected to the stomodeum (a paternal characteristic). However, in the reciprocal cross between A. latespinosus eggs and A. scoparius sperm, barrel-shaped larvae resembling those of A. latespinosus were produced, in addition to bipinnaria-like larvae, some with functional digestive systems and some with nonfunctional ones. Juveniles were produced from all types of crosses. 18S rDNA was used as a gene marker in cycle sequencing analysis to investigate the genetic features of these juveniles. The sequences of juveniles from bipinnaria-like larvae showed double-peak nucleotide signals, indicating a biparental genome. On the other hand, juveniles from barrel-shaped larvae from A. latespinosus eggs and A. scoparius sperm showed the same sequence as A. latespinosus juveniles. This suggests that bipinnaria-like larvae of both crosses are always hybrids, whereas barrel-shaped larvae develop parthenogenetically.     

Lactate dehydrogenase ontogeny,paternal gene activation,and tetramer assembly in embryos of brook trout,lake trout,and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.This study was supported by National Science Foundation Grant GB-7271.     

Genotypic and Phenotypic Characterization of the Drosophila Melanogaster Olfactory Mutation Indifferent

Two Drosophila melanogaster third chromosomes carrying the EMS-induced mutations IndifferentA (IndfA) and IndifferentB (IndfB), previously isolated from larvae showing an anosmia when stimulated with nonanol, were recombined with a multi-marked chromosome in order to localize the mutant character(s). Recombinant strains were tested for their larval olfactory responses and classed as either mutant or wild type; both Indf characters were found to be located on the right arm of the chromosome, between ebony and claret. Deletion mapping suggests that the Indifferent wild-type character is a haplo-insufficiency and that IndfA and IndfB are located in cytological region 96A2-7. Deficiencies and both mutant strains were tested with 14 closely related odors (alcohols, acetates, acids and methyl esters, between eight and 10 carbons long). When stimulated with methyl octanoate, IndfA and IndfB appeared recessive; noncomplementation was observed for this phenotype in IndfA/IndfB hybrids indicating that the two characters are allelic. The overall responses of IndfA, IndfB and the deficiencies indicate that Indf is involved in processing organic odors of between eight and 10 carbons in length.     

What Can Molecular and Morphological Markers Tell Us About Plant Hybridization?

The study of hybrids and their evolutionary significance is often based on a number of tacit assumptions regarding character expression in hybrids. This article examines morphological, chemical, and molecular character expression in hybrids to determine whether traditionally recognized properties of hybrid plants, such as hybrid intermediacy and character coherence, are actually supported by empirical evidence, and also examines the impact of hybrids on phylogenetic analyses. We show that hybrids are a mosaic of both parental and intermediate morphological characters rather than just intermediate ones, and that a large proportion of first (64%) and later generation hybrids (89%) exhibit extreme or novel characters. Chemical character expression in hybrids is more predictable, with predominantly additive or complementary expression for both first generation hybrids (68%) and hybrid taxa (54%). Likewise, the genetic basis, and thus the expression of molecular characters, is well-worked out and predictable, although non-Mendelian inheritance has been reported in a few instances for molecular markers as well.

There is even less empirical support for the concept of character coherence than hybrid intermediacy. Although morphological character coherence has been reported in both artificial and natural hybrid populations, it appears to be the exception rather than the rule. This idiosyncratic relationship among morphological characters is shown to be the norm for the relationship of morphological to molecular characters, as well as for the relationship among molecular characters.

Hybrids are shown to have little impact on the topology of nonhybrid taxa in phylogenetic trees. However, the expression of primitive vs. derived character states, the placement of hybrids in cladograms, the number of equally parsimonious trees produced, and the effects of hybrids on phylogenetic topology do not appear to be predictable. Thus, cladistic identification of hybrid taxa is difficult and may not be possible based on phenotypic data, regardless of the analytical tools used.     

Hybridization in coral reef fishes: introgression and bi-directional gene exchange in Thalassoma (family Labridae)

Hybrids in coral reef fishes have traditionally been described based on external features using meristic characters and colouration to identify putative parental contributors. This study utilised molecular genetic techniques to verify hybrid status and identify putative parental species for five hybrid specimens (Labridae: Thalassoma) collected from Holmes Reef in the Coral Sea. Phylogenetic analyses support hybrid origins of the specimens. Mitochondrial COI gene, nuclear S7 (intron 1) and nuclear copy of mitochondrial (NUMT) D-loop region corroborate the identity of T. quinquevittatum as the maternal and T. jansenii as the paternal contributor. Backcrossing to parental species by hybrids and bi-directional gene exchange between the Holmes Reef populations of T. jansenii and T. quinquevittatum was detected, suggesting that hybrids are fertile and able to reproduce successfully. F(1) hybrids display a mixture of the colouration attributes of the two parental species, but subsequent backcrossed individuals were unrecognisable as hybrids and displayed colouration of either parental species. A large numerical imbalance exists between the putative parental species at Holmes Reef, with T. quinquevittatum outnumbering T. jansenii by approximately 25:1. In this case study, hybridization appears to be driven by ecological rather than evolutionary factors.     

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs'' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.     

Assessment of Napier millet (Pennisetum purpureumx P. glaucum) and sorghum (Sorghum bicolor) trap crops for the management of Chilo partellus on maize

Two Napier millet (Pennisetum purpureumxP. glaucum) hybrids, namely PBN 83 and PBN 233 and one sorghum (Sorghum bicolor) variety, SL 44, were assessed for their potential role as a trap crop in the management of the stem borer, Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) on maize. Oviposition preference and larval survival and development were determined for different test plants under laboratory and screen house conditions. Further, field dispersal of C. partellus larvae was assessed between Napier millet and maize crops. Results from no-choice and dual-choice tests indicated that Napier millet hybrids were preferred for oviposition over maize by C. partellus moths. Sorghum was, however, not preferred over maize in this respect. Napier millet hybrids were poor larval hosts, and a rapid decline in larval numbers was noticed within the first five days after hatching and virtually no larvae survived to pupation. Leaf area eaten by the borer larvae was significantly less on these hybrids than on maize or sorghum. Plant damage was more severe in maize and sorghum than Napier millet hybrids. No appreciable larval shift was noticed from Napier millet hybrids to the adjoining maize crop. The evaluated Napier millet hybrids, therefore, had potential for use as trap crop in C. partellus management. Sorghum, however, did not hold promise in this respect.     

Nuclear function during early development of remote fish hybrids

The participation of paternal genome was studied in the development of remote hybrids obtained as a result of artificial insemination of the loach (Misgurnus fossilis) eggs by the sperm of aquarial Cyprinids (Brachydanio rerio, Danio malabaricus, Barbus tetrazona, Razbora heteromorpha, Carassius auratus) and Cobitids (Acanthophtalmus kuhlii). The hybrids obtained differed at the stage of hatching both from each other and from the loach by some morphological features. To study the function of heterologous nuclei, haploid nucleocytoplasmic hybrids were obtained by means of chromosome inactivation in the loach eggs by heavy doses of X-rays. The participation of paternal genome in development was estimated by comparison of the curves of viability of diploid and haploid hybrids with those of diploid, haploid and "anuclear" loach embryos. Patterns of mortality of embryos and larvae in each hybrid combination (percentage, stage) suggest the functioning of paternal genome already at the early stages of development. The activity of hybrid and heterologous nuclei was also estimated by the onset and the intensity of morphogenetic function which was determined by the time of embryonic death following nuclear inactivation at different stages. The onset of nuclear function in all hybrids coinsides with that in the loach, except B. rerio in which it occurs somewhat earlier. The data obtained prove the participation of paternal genes in development and maintenance of viability of embryos at all developmental stages beginning from the early ones (blastula).     

Morphologic comparisons of hatchery-reared specimens of Scaphirhynchus albus, Scaphirhynchus platorynchus, and S. albus × S. platorynchus hybrids (Acipenseriformes: Acipenseridae)

Extensive habitat modifications within the Mississippi and Missouri rivers have presumably interfered with the reproductive isolating mechanisms between the endangered pallid sturgeon, Scaphirhynchus albus, and the sympatric shovelnose sturgeon, Scaphirhynchus platorynchus, causing hybridization between these two species. Several character indices were developed to assist fisheries biologists in identifying specimens of S. albus, S. platorynchus, and their putative hybrids. The indices have numerous assumptions, including that pure strains of both parental species are within the sample analyzed and that hybrids are morphologically intermediate relative to their parents. If these indices have produced inaccurate identifications, then previous work on Scaphirhynchus studies in the Mississippi and Missouri rivers are questionable, including status surveys, captive propagation efforts, or the harvesting of tissues for genetic studies. In this study we tested indices by examining progeny of ‘known’ pallid, shovelnose, and hybrid sturgeon propagated, raised, and preserved at hatcheries. These 60 specimens [78–600 mm standard length (SL)] were propagated with breeding stock from the upper Missouri River drainage, where hybridization between these two species presumably does not occur. Existing indices did not correctly identify small (<250 mm SL) or a combination of small and large (>250 mm SL) sizes of S. albus, S. platorynchus, and their hybrids. Indices worked fairly well in identifying large S. platorynchus, but not in differentiating large S. albus from hybrids. We used principal components analysis (PCA) as an alternative approach to character indices. No a priori knowledge of the identity of the specimen is required with this multivariate technique, which avoids potential circular reasoning present in indices. We were able to completely or almost completely separate both sturgeon species and their hybrids by extracting principal components from a correlation matrix of 13 meristic characters in a standard PCA and extracting size‐corrected principal components from a covariance matrix of 51 morphometric variables using a sheared PCA. Additionally, we demonstrated that first generation hybrids were intermediate with respect to their parental species. Multivariate analyses with a reduced character set of six meristic and 12 morphometric variables also led to accurate and reliable specimen identification. Head spines and numerous qualitative characters are also extremely useful in differentiating between Scaphirhynchus species and their hybrids. In addition to all morphometric characters, some meristic characters and the degree of head spine fusion vary significantly with SL of sturgeons. Recording appropriate data from released specimens, including photovouchers, and making this information available is essential for researchers to have any scientific or legal basis for genetic or any other studies involving these sturgeons.     

Triple hybrids obtained by artificial hybridization of the Russian sturgeon Acipenser gueldenstaedtii with the hybrid of the starred sturgeon A. stellatus and the great sturgeon A. huso (Acipenseridae): The kind of inheritance of some morphological characters and fertility of the parental hybrid form

Results are described of experimental crossing for obtaining of triple hybrids of the Russian sturgeon Acipenser gueldenstaedtii, the great sturgeon A. huso, and the starred sturgeon A. stellatus and of comparative analysis of some quantitative morphological characters in these hybrids, in the progeny of parental species and of reciprocal hybrids of starred sturgeon and great sturgeon. By most investigated characters, in triple hybrids, the states of the paternal species (Russian sturgeon) are inherited, by some other characters-of great sturgeon-the state of starred sturgeon is not inherited. Therefore, on the basis of external appearance, they may be identified as the hybrid of Russian sturgeon with great sturgeon. The observed segregation by the form of gill rakers into paternal, maternal, or intermediate variants leads to a conclusion of nonmonogenous inheritance of a wide form and a conical form of gill rakers in sturgeons. On the whole, the obtained results clearly demonstrate how, due to effects of matrocliny and partocliny, and different combination of dominant and recessive alleles of the genes determining the diagnostic characters, the hybrid individuals may acquire a different extent of similarity and differences from the parental species. The investigated triple hybrids are an interesting example of mutual leveling of divergent traits in the course of combination of different genomes within limits of one closely related Atlantic group of sturgeons. As a result, a certain averaged habitus is formed which might take place also in case of formation of polyploidy species in the process of evolution.     

The question of fluctuating asymmetry in the blacklegged tick Ixodes scapularis (Acari: Ixodidae)

An analysis of fluctuating asymmetry was conducted on populations of the blacklegged tick Ixodes scapularis. The eight groups used in this study consisted of larvae and nymphs and males and females from the states of Minnesota, Massachusetts, Maryland, Missouri, North Carolina and Georgia and the F1 progenies of reciprocal crosses between ticks from Massachusetts and Georgia. Measurements included 16 larval, 19 nymphal, ten female and 12 male bilateral characters. Only five differences between the right and left bilateral characters had normal distributions with means of zero and differences in variances between the groups. These five characters included three setal lengths of the larvae, the spiracular plate length of females and the coxa I internal spur widths of males. Bivariate plots of character size ((R+L)/2) and asymmetry (R-L) showed no correlation. In the spiracular plate lengths of females and one of the setal lengths, ticks from Massachusetts had significantly less within-group variance than all the other groups. The only character in which fluctuating asymmetry was observed was the coxa I internal spur width of males, in which ticks from Minnesota, Missouri and North Carolina had significantly greater variance than the remaining groups; fluctuating asymmetry in this character may be explained by sexual selection. The cross progeny did not demonstrate any fluctuating asymmetry, as would be expected if the northern and southern forms of I. scapularis were true species. The virtual lack of fluctuating asymmetry in the characters used in this study further supports the conclusions of other studies which concluded that I. scapularis is a species with clinal variation and a broad geographic distribution.     

Predicting hybrid value in bread wheat: biometric modelling based on a ”top-cross” design

Multi-environmental experimentation made it possible to compare 311 hybrids and their 59 parental lines for five characters: yield, two yield components (number of grains per m² and 1000-grain weight), height and date of heading. At all three sites, average high-parent heterosis exceeded 6%, and our results confirm that hybrids in general head as early as the earlier parent does and are as high as the taller parent. For the five characters studied ecovalences were significantly lower in the F1 than in the parents, indicating a higher level of homeostasis for hybrids. The experimental model, based on a ”top-cross” design comprised of 55 lines used as females and four testers, made it possible to implement different hybrid-value prediction models. For yield, by far the most important character to predict, the predictor based on the general combining ability and the per se value of the parents, appears to be the most interesting. The prediction remains, however, imperfect as specific combining ability (SCA) has a strong impact for this character. Attempting to take account of SCA in the prediction, by using a multiplicative model for ”male×female” interaction modelling, did not prove convincing. Received: 15 February 1999 / Accepted: 25 July 1999